Copper is an essential transition metal frequently increased in cancer known to strongly influence essential cellular processes. Targeted therapy protocols utilizing both novel and repurposed drug agents initially demonstrate strong efficacy, before failing in advanced cancers as drug resistance develops and relapse occurs. Overcoming this limitation involves the development of strategies and protocols aimed at a wider targeting of the underlying molecular changes. Receptor Tyrosine Kinase signaling pathways, epigenetic mechanisms and cell metabolism are among the most common therapeutic targets, with molecular investigations increasingly demonstrating the strong influence each mechanism exerts on the others. Interestingly, all these mechanisms can be influenced by intracellular copper. We propose that copper chelating agents, already in clinical trial for multiple cancers, may simultaneously target these mechanisms across a wide variety of cancers, serving as an excellent candidate for targeted combination therapy. This review summarizes the known links between these mechanisms, copper, and copper chelation therapy.
Different carbon nanostructures have been explored as functional materials for the development of effective nanomaterials in cancer treatment applications. This review mainly aims to discuss the features, either strength or weakness, of carbon nanohorn (CNH), carbon conical horn-shaped nanostructures of sp2 carbon atoms. The interest for these materials arises from their ability to couple the clinically relevant properties of carbon nanomaterials as drug carriers with the negligible toxicity described in vivo. Here, we offer a comprehensive overview of the recent advances in the use of CNH in cancer treatments, underlining the benefits of each functionalization route and approach, as well as the biological performances of either loaded and unloaded materials, while discussing the importance of delivery devices.
DIPG is an incurable pediatric brain cancer of the ventral pons characterized by its complex epigenetic profile. Up to 81% of patients present with mutation H3K27M, resulting in the global reduction of H3K27 trimethylation and increased H3K27 acetylation, deregulating gene expression through an aberrant pattern of epigenetic modification. These alterations affect many cellular and metabolic mechanisms, complicating the search for effective targeted therapeutic strategies. Copper is highly abundant in the pons and is essential for normal brain function and development. However, excess copper accumulation is implicated in several neurological diseases and cancers. Incidentally, recent investigations have indicated the H3-H4 dimer can interact with copper acting as a reductase enzyme. Copper chelation therapy is clinically approved for pediatric patients with Wilson’s Disease, improving their neurological symptoms, and is being trialed in several cancers. We therefore hypothesized copper chelation may represent an effective therapeutic strategy for DIPG. Copper chelator tetraethylenepentamine (TEPA) decreased cell growth and induced apoptosis in DIPG cell lines. To understand these results, unbiased RNA-seq and metabolomics analyses were performed, revealing downregulation of EZH2, DNMT1 and DNMT3B, upregulation of KDM6B and the disruption of key enzymes in the S-adenosylmethionine (SAM)-cycle. Importantly, TEPA downregulated SAM, which donates methyl groups for methylation, S-adenosylhomocysteine, its post-methylation product and α-ketoglutarate, a co-factor for KDM6B. Western blots confirmed the reduced expression of EZH2, DNMT1 and DNMT3B, with further blots examining the chromatin cell fraction revealing modulation of H3K27 trimethylation through copper or TEPA stimulation, and reduction of H3K27 acetylation by TEPA. Importantly, in vitro combinations with Panobinostat were synergistic, while in vivo investigations demonstrated TEPA improved survival in an orthotopic patient derived xenograft (PDX) model, showing complete tumor regression in 25% of treated mice. This study indicates a novel use for copper chelators as epigenetic drugs, and their potential as therapeutics for DIPG.
Copper is a trace element essential to cellular function with elevated levels implicated in cancer progression. Clinical trials using copper chelators are associated with improved patient survival, however, the molecular mechanisms by which copper depletion inhibits tumor progression are poorly understood. This remains a major hurdle to the clinical translation of copper chelators. Epithelial-mesenchymal transition (EMT) is often exploited by malignant cells to promote growth and metastasis. Transforming growth factor (TGF)-β is a master regulator of EMT and facilitates cancer progression through changes in the tumor and its microenvironment. Herein, we report that a reduction of copper with the chelating agent tetraethylenepentamine (TEPA) inhibited EMT in vitro in three diverse cancer cell types; human triple-negative breast cancer (TNBC), neuroblastoma (NB), and diffuse intrinsic pontine glioma (DIPG) cell lines. Single-molecule imaging demonstrated EMT markers including Vimentin, β-catenin, ZEB1, and p-SMAD2 had increased expression with copper treatment and this pro-mesenchymal shift was rescued by the addition of TEPA. Moreover, SNAI1, ZEB1, and p-SMAD2 demonstrated increased accumulation in the cytoplasm after treating with TEPA. Transcriptomic analyses revealed a significant downregulation of the EMT pathway, including canonical (TGF-β/SMAD2&3) and non-canonical (TGF-β/PI3K/AKT and TGF-β/RAS/RAF/MEK/ERK) TGF signaling pathways. Matrix metalloproteinases MMP-9 and MMP-14 proteins which activate latent TGF-β complexes were also downregulated by TEPA treatment. These molecular changes are consistent with reduced plasma levels of TGF-β we observed in cancer models treated with TEPA. Importantly, copper chelation reduced metastasis to the lung in a TNBC orthotopic syngeneic mouse model. Our studies suggest copper chelation therapy can be used to inhibit EMT-induced metastasis by targeting TGF-β signalling. Because on-target anti-TGF-β therapies are failing in the clinic, copper chelation presents itself as a potential therapy for targeting TGF-β in cancer.
DIPG is an incurable and inoperable pediatric brain cancer in the ventral pons characterized by its complex mutational profile. Epigenetic mutations to H3K27M are seen in up to 81% of patients, resulting in the global loss of the repressive H3K27 trimethylation marker, increasing chromatin accessibility and deregulating gene expression. However, these epigenetic changes are often partnered by secondary mutations, with activating mutations and copy number gains observed in a wide variety of RTK genes and their associated signaling pathways. The combination of these two mutational paradigms results in the broad alteration of cellular and metabolic mechanisms, complicating the search for effective targeted therapeutic strategies. Copper, a metal ion essential for normal cellular function is known to be highly expressed in the pons, and influence ERK and PI3K/AKT signaling. Importantly, excess copper accumulation has been implicated in the pathogenesis of multiple neurological diseases and cancers. Copper chelation therapy is a clinically approved strategy for pediatric patients with Wilson’s Disease, known to improve their neurological symptoms, and is under investigation for use in a number of cancers. Therefore, we hypothesized copper chelation may be an effective therapeutic strategy for DIPG. Cytotoxicity assays using copper chelator tetraethylenepentamine (TEPA) demonstrated millimolar efficacy and synergized with mTOR inhibitor everolimus in a panel of DIPG cell lines, with H3-K27M mutant cell lines found to be more sensitive. Western blots were used to identify copper integrated pathways, with DIPG cell lines stimulated with sub-lethal copper concentrations demonstrating increased phosphorylated expression of ERK1/2, ERK5, AKT and p70SK61. TEPA reduced the phosphorylation of these markers compared to controls. Importantly, epigenetic mechanisms were sensitive to copper, as both copper stimulation and TEPA modulated H3K27 trimethylation, and TEPA decreased pro-expression H3K27 acetylation. In further blots examining epigenetic regulators, TEPA reduced the expression of EZH2, DNMT3B and DNMT1. RNA-sequencing analysis revealed TEPA downregulated genes involved in S-adenosylmethionine production metabolism, DNA regulation, cell cycle and mTORC signaling and specifically, EZH2, DNMT3B and DNMT1 expression. In vivo investigations demonstrated TEPA’s ability to improve survival in an orthotopic xenograft model, and via xenogen luminescence imaging complete tumor regression in 25% of treated mice. This study indicates copper directly impacts epigenetic and RTK mechanisms, and that their targeting through copper chelation agents represents a potential therapeutic strategy for DIPG, both as a single agent and in combination. Citation Format: Filip Michniewicz, Federica Saletta, Tayla Watkinson, Daniele Mercatelli, Federico M. Giorgi, Jessica Bell, Maria Tsoli, David Ziegler, Orazio Vittorio. Using copper chelating agents to simultaneously target epigenetic mechanisms and receptor tyrosine kinase (RTK) signaling in diffuse intrinsic pontine glioma (DIPG) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5716.
DIPG is a universally fatal pediatric brain cancer. Receptor tyrosine kinase (RTK) pathway alterations are among the defining characteristics in many patients. Copper is a transition metal essential for cellular signaling, known to impact PI3K/AKT and MAPK/ERK pathways. Copper chelating agents are clinically approved for use in children with Wilson’s Disease, documented to reduce brain copper levels and are cited as potential cancer therapeutics. Due to copper’s wide cellular integration, we propose that targeting copper in DIPG through use of copper chelators is a viable therapeutic strategy and are strong candidates for combination therapy. Cytotoxicity assays performed in a panel of DIPG cell lines using copper chelator tetraethylenepentamine (TEPA) demonstrated a millimolar range of efficacy. To identify copper integrated pathways, western blots were performed on DIPG cell lines dosed with sub-lethal copper concentrations, which increased phosphorylated expression of AKT, ERK1/2, ERK5 and STAT3. Conversely, western blots performed after TEPA treatment demonstrated reduced phosphorylated expression of all these proteins compared to controls. Western blots investigating TEPA in combination with Everolimus and Trametinib demonstrated synergistic targeting of these proteins. Our results indicate that adding copper in the culture media initiated two RTK-mediated downstream signal transductions, including AKT and ERK and additionally STAT signaling. The use of copper chelator TEPA affected copper homeostasis and reduced DIPG cell proliferation. Our study proposes copper plays an important role in RTK-mediated signaling promoting DIPG proliferation. This implies that reducing copper with clinically available chelation agents can represent a potential anti-cancer treatment for DIPG.
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